Polymer particles containing an entrained solute, e.g., dye, are widely used as markers for biomolecules and as internal reference and calibration standards for assay detection methods such as flow cytometry. Four general methods have been described in the prior art for producing fluorescent polymer particles: (A) copolymerization of dye and monomer; (B) partitioning of water-soluble or oil-soluble dyes into preformed particles; (C) surface functionalization of preformed particles; and (D) encapsulation of dye droplets. In addition, polymerization methods also have been used to prepare core-shell particles, that is, microparticles comprised of a polymer core and a polymer shell.
A. Copolymerization Based Methods
Fluorescent microparticles may be synthesized by polymerization of monomeric units to form microparticles in the presence of fluorescent dyes. U.S. Pat. No. 4,326,008 to Rembaum (1982) describes the synthesis of fluorescent microparticles by copolymerization of functionalized acrylic monomer with a polymerizable fluorescent comonomer. The method generally requires a polymerizable dye molecule. Such methods, generally suffer from the drawback of possible inhibition of polymerization by the fluorescent dye and/or bleaching of the fluorescence by the reactive constituents of the polymerization reaction.
U.S. Pat. No. 4,267,235 to Rembaum (1981) describes the synthesis of polygluteraldehyde microspheres using suspension polymerization. Cosolubilized fluorescein isothiocyanate (FITC) is used to create fluorescent microspheres. Suspension condensation polymerization of the monomer with cosolubilized dye molecules, while largely circumventing dye destruction and polymerization inhibition, generates a broad particle size distribution and hence is not a suitable route for the production of monodisperse fluorescent microspheres.
U.S. Pat. No. 5,073,498 to Schwartz et al. (1991) describes a process for making fluorescent microparticles by seeded polymerization. One or more hydrophobic fluorescent dyes are dissolved in a solution containing monomer and initiator. The solution is added to pre-swollen microparticles. The patent discloses methods permitting the introduction of three different dyes into a particle. The method suffers from the drawback of possible inhibition of polymerization by the fluorescent dye, or conversely the bleaching of the fluorescence by the polymerization process.
Multi-stage emulsion polymerization has been employed to prepare core-shell particles without surface functional groups. U.S. Pat. No. 5,952,131 to Kumaceheva et al. discloses a method for preparing stained core-shell particles. The method is based on multiple stages of semi-continuous polymerization of a mixture of two monomers (methyl methacrylate and ethylene glycol dimethacrylate) and a fluorescent dye (4-amino-7-nitrobnezo-2-oxa-1,3 diazol-labeled methyl methacrylate). The particles are then encapsulated with an outer shell by copolymerization of methyl methacrylate and butylmethacrylate in the presence of chain transfer agent, dodecyl mercaptan. Kumaceheva et al. do not prepare and do not have as an object the inclusion of surface functional group core-shell polymer product.
U.S. Pat. No. 4,613,559 to Ober et al. discloses a method for preparing colored toner by swelling. Polystyrene particles (5.5 micron) are prepared by dispersion polymerization of styrene in the presence of ethanol, poly(acrylic acid), methylcellosolve and benzoyl peroxide. Swelling is performed by dispersing the polystyrene in an aqueous solution of sodium dodecyl sulfate and acetone. Colored particles are obtained by adding an emulsified dye solution (Passaic Oil Red 2144 in methylene chloride emulsified with an aqueous solution of sodium dodecylsulfate) to the particle dispersion.
Polymerization methods have been employed to prepare core-shell particles containing surface functional groups. U.S. Pat. No. 5,395,688 to Wang et al. discloses magnetically-responsive fluorescent polymer particles comprising a polymeric core coated with a layer of polymer containing magnetically-responsive metal oxide. The final polymer shell is synthesized with a functional monomer to facilitate covalent coupling with biological materials. The procedure of Wang et al. is based on three steps: (1) preparation of fluorescent core particles; (2) encapsulation of metal oxide in a polystyrene shell formed over the fluorescent core by free radical polymerization in the absence of emulsifier but with an excess of initiator; and (3) coating of the magnetic fluorescent particles with a layer of functional polymer. The functional polymer has carboxyl, amino, hydroxy or sulfonic groups. Wang et al. do not describe a method for obtaining the colored core and also does not address the problem of destruction of dye during the free radical polymerization process.
U.S. Pat. No. 4,829,101 to Kraemer et al. discloses two-micron fluorescent particles obtained by core-shell polymerization. The core is obtained at 80° C. by polymerizing a mixture of isobutyl methacrylate, methyl methacrylate and ethylene glycol dimethacrylate via ammonium persulfate initiation. A shell is synthesized over the core by semi-continuously adding, in a first step, a mixture of the same monomers containing a fluorescent dye (fluoro-green-gold). Through the end of the reaction, two different monomer mixtures are added over a one hour period: a first mixture containing methyl methacrylate, ethylene glycol-bis-(methacrylate) and glycidyl methacrylate, and a second mixture containing methacrylamide and initiator. The polymerization is initiated with 4,4′-azobis-(cyanovaleric acid).
Okubo et al., Colloid Polym. Sci. 269:222-226 (1991), Yamashita, et al., Colloids and Surfaces A., 153:153-159 (1999), and U.S. Pat. No. 4,996,265 describe production of micron-sized monodispersed polymer particles by seeded dispersion polymerization. Polymer seed particles are pre-swelled with large amounts of monomer prior to seeded polymerization. The swelling is carried out by slow, continuous, drop-wise addition of water to an ethanol-water mixture containing the seed particles, monomers, stabilizer and initiator. The addition of water decreases the solubility of the monomer in the continuous phase, leading to precipitation and subsequent absorption of monomer onto or into the seed polymer particles. The monomer absorbed into the seed polymer particle is then polymerized to produce large monodispersed polymer particles.
B. Partitioning of Water-Soluble or Oil-Soluble Dyes
Fluorescent particles can be produced by permitting dye molecules to partition into pre-swollen microparticles according to a technique originally described by L. B. Bangs (Uniform Latex Particles; Seragen Diagnostics Inc., 1984, p. 40). The process involves dissolution of a dye molecule or mixture of dye molecules in a solvent or solvent mixture of choice containing polymer microparticles. Absorption of the solvent by the microparticles leads to swelling, permitting the microparticles to absorb a portion of the dye present in the solvent mixture. The staining process is usually terminated by removing the solvent. The level of dye partitioning is controlled by adjusting the dye concentration, and in the case of a plurality of dyes, the relative abundance of individual dyes. Microparticles stained in this manner are quite stable and uniform. However, in many cases, depending on the choice of solvent system, a large dye excess is required to attain the desired partitioning, leading to significant loss of expensive dye material.
U.S. Pat. No. 5,723,218 to Haugland et al. (1998), U.S. Pat. No. 5,786,219 to Zhang et al. (1998), U.S. Pat. No. 5,326,692 to Brinkley et al. (1994) and U.S. Pat. No. 5,573,909 to Singer et al. (1996) describe protocols for producing various fluorescently-colored particles by swelling and dye partitioning in organic solvent and organic solvent mixtures. Various types of fluorescent particles, for example, fluorescent particles containing multiple dyes, particles exhibiting controllable and enhanced Stokes shifts, and particles displaying spherical zones of fluorescence, are described.
International patent application WO 99/19515 of Chandler et al. (1997) describes an improved method for the production of a series of ratiometrically-encoded microspheres with two dyes. A protocol for the production of 64 different encoded microspheres is reported. A swelling bath composition using a mixture of an organic solvent and alcohol (under anhydrous conditions) also is disclosed.
U.S. Pat. No. 5,266,497 to Matsudo et al. (1993) describes a method for generating a dye-labeled polymer particle which uses a hydrophobic dye dissolved in an organic solvent emulsified in water. The dyed particles were used for immuno-chromatographic purposes.
U.S. Pat. No. 4,613,559 to Ober et al. (1986) describes the synthesis of colored polymer particles using oil-soluble dyes. The disclosed method uses an emulsion of a dichloromethane dye solution in a water and acetone mixture for coloring the particles.
C. Functionalization of Internal or External Microparticle Surfaces
Production of fluorescent particles by surface functionalization involves the covalent attachment of one or more dyes to reactive groups on the surface of a preformed microparticle. This leaves the dye molecules exposed to the environment, which can hasten the decomposition of the dye. In addition, surface functionalization often renders a particle surface very hydrophobic, inviting undesirable non-specific adsorption and, in some cases, loss of activity of biomolecules placed on or near the particle surface. These problems can be circumvented by attaching a stained small particle, in lieu of a dye molecule, to the surface of a carrier particle. The efficacy of this method in generating large sets of encoded particles from a small number of dyes (ratio encoding) is unclear.
U.S. Pat. No. 4,487,855 to Shih (1984), U.S. Pat. No. 5,194,300 to Cheung (1993) and U.S. Pat. No. 4,774,189 to Schwartz (1988) disclose methods for preparation of colored or fluorescent microspheres by covalent attachment of either one or a plurality of dyes to reactive groups on the preformed particle surface. Battersby et al., “Toward Larger Chemical Libraries: Encoding with Fluorescent Colloids in Combinatorial Chemistry” J. Am. Chem. Soc. 2000, 122, 2138-2139; Grondahl et al., “Encoding Combinatorial Libraries: A Novel Application of Fluorescent Silica Colloids”, Langmuir 2000, 16, 9709-9715; and U.S. Pat. No. 6,268,222 to Chandler et al. (2001) describe a method of producing fluorescent microspheres by attaching to the surface of a carrier microparticle a set of smaller polymeric particles that are stained.
D. Encapsulation Methods
Formation of fluorescent particles by encapsulation utilizes a solution of a preformed polymer and one or more dyes. In one approach, the solution is dispensed in the form of a droplet using a vibrating nozzle or jet, and the solvent is removed to produce polymer particles encapsulating the dye. This process requires specialized process equipment and displays only limited throughput. Alternatively, a polymer dye mixture is emulsified in a high-boiling solvent and the solution is evaporated to yield polymer-encapsulated dye particles. This process often generates non-spherical particles with broad size distribution.
U.S. Pat. No. 3,790,492 to Fulwyler et al (1974) discloses a method to produce uniform fluorescent microspheres from a pre-dissolved polymer and dye solution using a jet. U.S. Pat. No. 4,717,655 to Fulwyler et al. (1988) discloses a process which includes two dyes in pre-designated ratios in a polymer microparticle to produce five distinguishable two-color particles.
The various prior art methods of producing fluorescent microparticles suffer from certain disadvantages. Where strong swelling solvents are used, the microparticles must be cross-linked to prevent them from disintegrating and deforming in the dye solution. This constraint represents a severe limitation since the majority of dyes require for their dissolution at any reasonable concentration solvent systems in which most polymer particles of interest, notably polystyrene particles, also will dissolve. These considerations have restricted the application of solvent swelling in the prior art to chemically stabilized (“cross-linked”) microparticles. This restriction introduces additional difficulty and cost in microparticle synthesis; highly cross-linked particles are often very difficult to synthesize. Also, restriction to cross-linked particles limits the degree of microparticle swelling and thus the degree of dye incorporation. Specifically, the application of solvent swelling protocols of the prior art conducted on cross-linked microparticles generally limits penetration of the dye to the outer layer of the microparticle, thereby precluding uniform staining of the entire interior volume of individual particles and generally also precluding the realization of high levels of dye incorporation. What is needed is a process that can utilize non-cross-linked, as well as cross-linked, particles. What is needed is a method that will provide dye-loaded non-cross-linked polymer microparticles, which may be used, for example, to prepare libraries of dyed microparticles having containing different dyes and/or different dye amounts.
The degree of particle swelling in prior art solvent swelling-based methods of dye incorporation determines the rate of dye transport into the particles. Diffusion barriers lead to non-uniform dye distribution in the microparticles. For this reason, intense micro-mixing (brought about by either efficient mechanical mixing or by sonication) is required in order to produce uniformly stained populations of microparticles. These vigorous mixing procedures, while effective for laboratory scale preparation, are not easily adapted to larger scales. For example, sonication often requires specialized equipment such as probe sonicators, and limits the parallel completion of multiple staining reactions. What is needed is a dyed particle manufacturing process that requires less vigorous mixing or no mixing, and permits parallel staining reactions to be performed.
Microparticles stained by prior art swelling methods are vulnerable to subsequent exposure to solvents that may cause substantial loss of dye and may preclude the implementation of protocols providing for multiple sequential dye incorporation steps.
In the prior art methods, the degree of dye partitioning into the polymer matrix is controlled by explicit variation of the initial concentration of dye in the dye solution. This approach, while permitting the realization of multiple, distinct levels of dye inclusion, suffers from a number of disadvantages. For example, high levels of staining frequently are not attainable because of the limited solubility of the dye in the bath. Even when solubility is not an issue, the low partition coefficients of many dyes requires a large excess of dye in solution introducing the risk of deleterious effects on subsequent bioanalytical assays. In fact, when carboxylate-modified beads are prepared by prior art solvent-swelling methods, the carboxyl function may become inoperative, and may be no longer available for functionalization by covalent coupling to other chemical groups. In addition, valuable dye material is lost in significant quantities. What is needed is a process for preparing stained microparticles, and fluorescent microparticles in particular, that achieves dye incorporation even from poorly soluble dye/solvent formulations. What is needed is a process that allows for precise control of the solute (dye) loading level in polymer microparticles during the staining process.